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WO2011150061A1 - Peptides de liaison au récepteur du facteur 1 de croissance de type insuline - Google Patents

Peptides de liaison au récepteur du facteur 1 de croissance de type insuline Download PDF

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Publication number
WO2011150061A1
WO2011150061A1 PCT/US2011/037904 US2011037904W WO2011150061A1 WO 2011150061 A1 WO2011150061 A1 WO 2011150061A1 US 2011037904 W US2011037904 W US 2011037904W WO 2011150061 A1 WO2011150061 A1 WO 2011150061A1
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WIPO (PCT)
Prior art keywords
polypeptide
seq
polypeptides
nos
sequence shown
Prior art date
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PCT/US2011/037904
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English (en)
Inventor
Michael Diem
Karen T. O'neil
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Janssen Biotech Inc
Original Assignee
Centocor Ortho Biotech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority to RS20150591A priority Critical patent/RS54278B1/sr
Priority to PL11787324T priority patent/PL2575846T3/pl
Priority to BR112012030084A priority patent/BR112012030084B1/pt
Priority to EA201291416A priority patent/EA023541B1/ru
Priority to UAA201214969A priority patent/UA107596C2/ru
Priority to DK11787324.0T priority patent/DK2575846T3/en
Priority to KR1020127033486A priority patent/KR101898502B1/ko
Priority to ES11787324.0T priority patent/ES2550040T3/es
Priority to EP11787324.0A priority patent/EP2575846B1/fr
Priority to MEP-2015-146A priority patent/ME02206B/fr
Priority to SI201130557T priority patent/SI2575846T1/sl
Priority to MX2012013710A priority patent/MX337134B/es
Priority to SG2012086617A priority patent/SG186068A1/en
Priority to AU2011258301A priority patent/AU2011258301B2/en
Priority to JP2013512202A priority patent/JP5864556B2/ja
Priority to CA2800744A priority patent/CA2800744C/fr
Priority to HRP20151084TT priority patent/HRP20151084T1/hr
Priority to CN201180025866.2A priority patent/CN103025341B/zh
Priority to HK13110609.7A priority patent/HK1183234B/xx
Application filed by Centocor Ortho Biotech Inc filed Critical Centocor Ortho Biotech Inc
Priority to NZ603611A priority patent/NZ603611A/en
Publication of WO2011150061A1 publication Critical patent/WO2011150061A1/fr
Priority to IL223030A priority patent/IL223030A/en
Anticipated expiration legal-status Critical
Priority to ZA2012/09689A priority patent/ZA201209689B/en
Priority to SM201500222T priority patent/SMT201500222B/xx
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • Insulin-like growth factor 1 receptor binding peptides Insulin-like growth factor 1 receptor binding peptides
  • the present invention relates to insulin-like growth factor 1 receptor binding peptides, polynucleotides encoding them, and methods of making and using the foregoing.
  • BBB blood-brain barrier
  • Therapeutics can cross the BBB using several pathways, including saturable transporter systems, adsorptive
  • transcytosis wherein the therapeutic to be transported is internalized by a cell in the BBB and routed to the abluminal surface for deposition into the brain intracellular fluid compartment, transmembrane diffusion wherein the therapeutic dissolves into the lipid bilayer which forms the membranes of the cells comprising the BBB, and extracellular pathways wherein the therapeutic exploits the residual leakiness of the BBB.
  • the current approaches to improve the transport of therapeutic agents across the BBB include ineffectiveness due to competition with the endogenous ligand, lack of transport of the therapeutic to brain parenchyma, and degradation of the therapeutics due to lysosomal targeting.
  • Fig. 1. shows binding of select phage lysates to IGF1R and IR.
  • Fig. 2. shows binding of peptide-AP fusions to IGF1R.
  • One aspect of the invention is an isolated polypeptide comprising a polypeptide having the sequence shown in SEQ ID NOs: 1-13.
  • Another aspect of the invention is an isolated
  • polynucleotide comprising a polynucleotide encoding the polypeptide comprising the amino acid sequence shown in SEQ ID NOs: 1-13.
  • Another aspect of the invention is an isolated
  • polynucleotide comprising a polynucleotide having the sequence shown in SEQ ID NOs: 14-26, or a complementary sequence thereof.
  • Another aspect of the invention is an isolated vector comprising a polynucleotide having the sequence shown in SEQ ID NOs: 14-26.
  • Another aspect of the invention is an isolated host cell comprising the vector of the invention.
  • Another aspect of the invention is an isolated fusion protein comprising a polypeptide having the sequence shown in SEQ ID NOs: 1-13 fused to a second polypeptide.
  • Another aspect of the invention is a method of
  • Another aspect of the invention is a method for delivery of a therapeutic agent across endothelial cells, comprising a. conjugating the therapeutic agent to a polypeptide comprising the polypeptide having the sequence shown in SEQ ID NOs: 1, 2, 4, 8, or 12 to form a conjugate; b. contacting the conjugate with the endothelial cells; and
  • polypeptide means a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as “peptides”. Polypeptides may also be referred as "proteins.”
  • polynucleotide means a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. Double and single-stranded DNAs and RNAs are typical examples of polynucleotides .
  • complementary sequence means a second isolated polynucleotide sequence that is antiparallel to a first isolated polynucleotide sequence and that comprises nucleotides complementary to the nucleotides in the first polynucleotide sequence.
  • complementary sequences are capable of forming a double-stranded
  • polynucleotide molecule such as double-stranded DNA or double-stranded RNA when combined under appropriate
  • vector means a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems.
  • Vector polynucleotides typically contain elements, such as origins of replication,
  • polyadenylation signal or selection markers that function to facilitate the duplication or maintenance of these
  • polynucleotides in a biological system examples include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector.
  • polynucleotides comprising a vector may be DNA or RNA molecules or hybrids of these.
  • expression vector means a vector that can be utilized in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.
  • blood-brain barrier refers to the barrier between the peripheral circulation and the brain and spinal cord which is formed by tight junctions within the brain capillary endothelial plasma membranes, creates an extremely tight barrier that restricts the transport of molecules into the brain, even molecules as small as urea having a molecular weight of 60 Da.
  • the blood- brain barrier within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina are contiguous capillary barriers within the central nervous system (CNS) , and are collectively referred to as the blood-brain barrier.
  • CNS central nervous system
  • antibody refers to a molecule specifically binding to an antigen, and includes dimeric, trimeric and multimeric antibodies, and chimeric, humanized and fully human antibodies.
  • an antibody may be a whole antibody or a functional fragment of an antibody molecule, such as a fragment retaining at least its antigen binding function, and include Fab, F(ab'), F(ab')2r scFv, dsFv, and diabodies .
  • antibody fragments may be obtained using proteolytic enzymes (e.g., a whole antibody is digested with papain to produce Fab fragments, and pepsin treatment results in the production of F(ab')2 fragments) .
  • Fully human monoclonal antibodies lacking any non-human sequences can be prepared from human immunoglobulin transgenic mice or from phage display libraries (Lonberg et al., Nature 368:856-859, 1994; Fishwild et al.
  • an antibody molecule or preparation "specifically binds" a given antigen when it binds this antigen with higher affinity and in a specific, as opposed to non-specific fashion, relative to a second non-identical antigen. Stated differently, the "specific binding" of an antibody molecule or preparation can be used to distinguish between two different polypeptides.
  • IGF1R insulin-like growth factor receptor 1
  • GenBank Acc . No. NP_000866 human IGF1R having an amino acid sequence shown in SEQ ID NO: 27.
  • the IGF1R pro-polypeptide is cleaved into an alpha and a beta chain to form a mature protein.
  • the alpha chain has amino acid residues 31-740 of SEQ ID NO: 27, and the beta chain has amino acid residues 741-1367 of SEQ ID NO: 27.
  • Soluble IGF1R or “sIGFIR” as used herein refers to the extracellular domain of the IGF1R (amino acids 31 - 932 of SEQ ID NO: 27) .
  • the soluble IGF1R may be an extracellular domain of an uncleaved pro-polypeptide, or an extracellular domain of a mature IGF1R (amino acid residues 31-740 forming the alpha chain, and amino acid residues 741-932 forming the extracellular portion of the beta chain) .
  • conjugate refers to a chimeric molecule comprising a peptide of the invention having an amino acid sequence shown in SEQ ID NOs : 1-13 and a
  • conjugating means that the therapeutic agent (s) and the peptides of the invention are physically linked by, for example, by covalent chemical bonds, physical forces such as van der Waals or hydrophobic interactions, encapsulation, embedding, or combinations thereof.
  • the therapeutic agent (s) and the peptides of the invention can be linked by chemical bonds through alcohol, acid, carbonyl, thiol or amine groups using well known chemical synthesis methods (see e.g. US Pat. Appl . No. US2010/0028370) .
  • the therapeutic agent can be connected to the peptides of the invention by a linker.
  • Exemplary linkers are glycine-rich linkers such as Gly 3 SerGly 3 Ser (SEQ ID NO: 28) or Gly 4 SerGly 4 SerGly 4 Ser (SEQ ID NO: 29) .
  • fusion protein refers to a polypeptide made up of two (or more) heterologous polypeptides which are not normally fused together in a single amino acid sequence. Fusion proteins may generally be prepared using either recombinant nucleic acid methods, i.e., as a result of transcription and translation of a recombinant gene fusion product, which fusion comprises a segment encoding a polypeptide of the invention and a segment encoding a heterologous polypeptide.
  • therapeutic agent refers to a molecule administered to induce a desired therapeutic effect in a subject.
  • a subject is a human or non-human animal, including a mammal or a primate.
  • exemplary therapeutic agents are proteins, antibodies, peptides, small molecules, or polynucleotides.
  • Therapeutic agents may also be toxins or radioisotopes, where the therapeutic effect intended is, for example, the killing of a cancer cell.
  • the present invention provides isolated polypeptides that bind IGF1R, polynucleotides encoding the polypeptides, vectors comprising the polynucleotides, isolated host cells, polypeptides obtainable from expression of the
  • polypeptides of the invention bind to the IGF1R, and are transcytosed across endothelial cells. Since IFG1R is expressed on the
  • the polypeptides of the invention can provide means for
  • One aspect of the invention is an isolated polypeptide comprising a polypeptide having the sequence shown in SEQ ID NOs: 1-13.
  • polypeptides of the invention may be produced by chemical synthesis, such as solid phase peptide synthesis, on an automated peptide synthesizer. Alternatively, the polypeptides of the invention can be obtained from
  • polypeptides of the invention can also be obtained by expression and isolation from cells harboring a nucleic acid sequence of the invention by techniques well known in the art, such as recombinant expression of easily isolated affinity labeled polypeptides. Those skilled in the art will recognize other techniques for obtaining the polypeptides of the invention.
  • Another aspect of the invention is an isolated fusion protein comprising a polypeptide having the sequence shown in SEQ ID NOs: 1-13 fused to a second polypeptide.
  • Such second polypeptide may be a leader or a secretory signal sequences .
  • Such second polypeptide may be a therapeutic agent fused to the peptides of the invention.
  • the therapeutic agent and the peptide of the invention can be fused to one another in a variety of ways.
  • the C-terminus or the N-terminus of the peptide of the invention may be directly linked to the N- terminus or the C-terminus, respectively of the therapeutic agent via an amide bond or a peptide linker.
  • Therapeutic agents may be linked to the peptide of the invention using chemical crosslinking well known in the art.
  • Another aspect of the invention is an isolated
  • polynucleotide comprising a polynucleotide encoding the polypeptides of the invention.
  • polynucleotides of the invention may be produced by chemical synthesis such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer.
  • polynucleotides of the invention may be produced by other techniques such as PCR based duplication, vector based duplication, or restriction enzyme based DNA manipulation techniques .
  • Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art .
  • polynucleotides of the invention may also comprise at least one non-coding sequence, such as transcribed but not translated sequences, termination signals, ribosome binding sites, mRNA stabilizing sequences, introns and
  • polyadenylation signals may also comprise additional sequences encoding additional amino acids .
  • additional polynucleotide sequences may, for example, encode a marker or tag sequence such as a hexa- histidine peptide (Gentz et al., Proc. Natl. Acad. Sci. (USA) 86:821-284, 1989) or the HA peptide tag (Wilson et al . , Cell 37:767-778, 1984) which facilitate the purification of fused polypeptides .
  • Exemplary polynucleotides are polynucleotides having a sequence shown in SEQ ID NOs : 14-26.
  • Another embodiment of the invention is a vector comprising an isolated polynucleotide having a sequence shown in SEQ ID NOs: 14-26.
  • the vectors of the invention are useful for maintaining polynucleotides, duplicating
  • Vectors may be chromosomal-, episomal- and virus-derived such as vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,
  • pseudorabies viruses picornaviruses and retroviruses and vectors derived from combinations thereof, such as cosmids and phagemids .
  • the vectors of the invention can be formulated in microparticles, with adjuvants, lipid, buffer or other excipients as appropriate for a particular application.
  • the vector is an expression vector.
  • Expression vectors typically comprise nucleic acid sequence elements that can control, regulate, cause or permit expression of a polypeptide encoded by such a vector. Such elements may comprise transcriptional enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate the expression of encoded polypeptides in a given expression system. Such expression systems may be cell-based, or cell-free systems well known in the art. Nucleic acid sequence elements and parent vector sequences suitable for use in the expression of encoded polypeptides are also well known.
  • An exemplary plasmid-derived expression vector useful for expression of the polypeptides of the invention comprises an E. coli origin of replication, a chloramphenicol acetyltransferase (CAT) gene, a bacteriophage T7 promoter, a pelB signal sequence, and a T7 terminator sequence.
  • CAT chloramphenicol acetyltransferase
  • Another embodiment of the invention is an isolated host cell comprising a vector of the invention.
  • Representative host cell examples include Archaea cells; bacterial cells such as Streptococci, Staphylococci, Enterococci, E. coli, Streptomyces, cyanobacteria, B. subtilis and S. aureus;
  • fungal cells such as Kluveromyces, Saccharomyces,
  • Basidomycete Candida albicans or Aspergillus
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1, Bowes melanoma and myeloma
  • plant cells such as gymnosperm or angiosperm cells.
  • the host cells in the methods of the invention may be provided as individual cells, or populations of cells. Populations of cells may comprise an isolated or cultured population of cells or cells present in a matrix such as a tissue.
  • a polynucleotide such as a vector
  • introduction of a polynucleotide, such as a vector, into a host cell can be effected by methods well known to those skilled in the art (Davis et al., Basic Methods in Molecular Biology, 2 nd ed., Appleton & Lange, Norwalk, CT, 1994;
  • microinjection cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
  • polypeptides or fragments of the invention for such purposes as enhancing substrate specificity, stability, solubility, and the like.
  • a modified polypeptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition. It is contemplated that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e., conservative mutations) will, in some instances but not all, not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic (aspartate,
  • glutamate glutamate
  • basic lysine, arginine, histidine
  • nonpolar alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • uncharged polar glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine
  • Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
  • the amino acid repertoire can be grouped as (1) acidic (aspartate, glutamate) ; (2) basic (lysine, arginine histidine) , (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine) , with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry, 2nd ed, WH Freeman and Co., 1981).
  • Whether a change in the amino acid sequence of a polypeptide or fragment thereof results in a functional homolog can be readily determined by assessing the ability of the modified polypeptide or fragment to produce a response in a fashion similar to the unmodified polypeptide or fragment using the assays described herein. Peptides, polypeptides or proteins in which more than one replacement has taken place can readily be tested in the same manner.
  • polypeptides of the invention can also be formulated in a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutically acceptable carrier or diluent e.g. 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well-known sterilization techniques (e.g., filtration).
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents.
  • concentration of the polypeptides of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities and other factors, according to the particular mode of administration selected.
  • the appropriate therapeutically effective dose can be determined readily by those of skill in the art. A determined dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician or other person skilled in the relevant art (e.g. nurse, veterinarian, or veterinary technician) during the treatment period.
  • polypeptides of the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with
  • Another embodiment of the invention is a method for expressing a polypeptide comprising the steps of providing a host cell of the invention; culturing the host cell under conditions sufficient for the expression of at least one polypeptide of the invention.
  • Host cells can be cultured under any conditions suitable for maintaining or propagating a given type of host cell and sufficient for expressing a polypeptide.
  • Culture conditions, media, and related methods sufficient for the expression of polypeptides are well known in the art.
  • many mammalian cell types can be aerobically cultured at 37 °C using appropriately buffered DMEM media while bacterial, yeast and other cell types may be cultured at 37 °C under appropriate atmospheric conditions in LB media.
  • the expression of a polypeptide can be confirmed using a variety of well known methods.
  • expression of a polypeptide can be confirmed using detection reagents, such as antibodies using for example FACS or immunofluorescent techniques, or using SDS-PAGE or HPLC .
  • Another aspect of the invention is a method for delivery of a therapeutic agent across endothelial cells, comprising: a. conjugating the therapeutic agent to a
  • polypeptide comprising the polypeptide having the sequence shown in SEQ ID NOs : 1, 2, 4, 8, or 12 to form a conjugate;
  • the polypeptides of the invention facilitate delivery of a therapeutic agent across endothelial cells through the binding of the polypeptides of the invention to an IGF1R.
  • the peptides are selected so that the conjugated therapeutic agent does not interfere with binding of the polypeptides of the invention to IGF1R.
  • the present invention describes conjugation of a protein having a molecular weight of approximately 100 kD (921 amino acids) to the polypepetides of the invention without loss of transcytosis activity.
  • polypeptides of comparable size are also likely to be successfully conjugated to the polypeptides of the invention and delivered across endothelial cell to the brain.
  • Delivery of the conjugate across the endothelial cells can be measured using well known in vitro or in vivo methods.
  • Exemplary in vitro measurements can be done using polarized monolayers of endothelial cells and measuring the
  • transcytosis of the conjugate using for example antibodies to the conjugate.
  • the present invention will now be described with reference to the following specific, non-limiting examples.
  • the pIX phage libraries displaying random peptides were generated according to methods described in US Pat. Appl. No. US2010/0021477, and used as a source of human IGF1R binding peptides .
  • This library was panned in solution against a biotinylated form of purified soluble IGF1R (sIGFIR) having a carboxy-terminal hexahistidine tag (R&D Systems, Minneapolis, MN) for three rounds. Biotinylation of sIGFIR was done using the EZ-Link No-Weigh Sulfo-NHS-LC-Biotin Microtubes (Pierce, Rockford, IL) . Because of the large size of sIGFIR ( ⁇ 330 kDa) , Tetralink Avidin beads were used for round 1 selection due to their ⁇ 10 fold greater binding capacity than Dynal magnetic beads.
  • Positive lysates were defined as clones with a sIGFIR specific signal that was three fold above the background of the negative control. A total of 13 clones with unique peptide sequences were obtained with confirmed binding to the sIGFIR (Table 1) . From these 13 clones, 3 (clones 5, 13 and 16) cross-reacted with the insulin receptor. The peptides from clones 5-14, 16 and 17 were cloned in- frame as peptide-Alkaline phosphatase-His6 (peptide-AP) fusion proteins into a modified pET20b+ vector having a Chloramphenicol acetyltransferase (CAT) gene cloned within.
  • CAT Chloramphenicol acetyltransferase
  • the resulting peptide-AP fusions were expressed in bacteria and purified using Ni-NTA (EMD Biosciences, Gibbstown, NJ) according to manufacturer' s instructions .
  • the amino acid sequence of the alkaline phosphatase used is shown in SEQ ID NO: 30.
  • the identified IFG1R binding peptides were cloned in- frame to the C-terminus of the protein G IgG domain (PG) in a modified pET17b vector (EMD Chemicals, Gibbstown, NJ) having a ligation independent cloning site (LIC) to generate PG- peptide fusions.
  • the IgG binding domain of protein G is stable and thus enabled easy purification of the fusion protein from bacterial lysates.
  • the amino acid sequence of the protein G IgG domain used is shown in SEQ ID NO: 31.
  • the PG-peptide fusions were expressed in bacteria upon 1 mM IPTG induction and purified using IgG Sepharose beads (GE
  • Rat Brain capillary endothelial cells were prepared as described (Perriere et al . , J. Neurochem. 93:279-289, 2005). Briefly, brains from 6-8 week old male Sprague Dawley rats were rolled on 3 M chromatography paper to remove the meniges, cut sagitally, and the white matter dissected leaving the cortices, which were then minced thoroughly.
  • the minced cortices were transferred to a 50 ml polypropylene conical tube with 20 ml DMEM supplemented with 39 units/ml DNase I (Worthington, Lakewood, NJ) and 0.7 mg/ml Collagenase type 2 (Worthington, Lakewood, NJ) at final concentration and incubated at 37°C with gentle mixing for 1.25 hrs. After a brief centrifugation the resultant pellet was resuspended in 20 ml 20% BSA (Sigma, St.
  • the cell pellet was resuspended in 10 ml complete rat brain microvascular endothelial cell growth media (DMEM, 20% Plasma Derived Serum (PDS) , 100 ug/ml Heparin, 2 mM L-Glutamine, 100 U/ml penicillin, 100 ⁇ g ml streptomycin, 0.25 ⁇ g/ l
  • DMEM complete rat brain microvascular endothelial cell growth media
  • PDS Plasma Derived Serum
  • Both chambers were supplemented with 4 ⁇ g/ml puromycin (Clontech, Mountain View, CA) and the plates were incubated at 37 °C, 5% C0 2 overnight. The next day the media was changed with fresh complete culture media with ⁇ g/ml puromycin and the cells were placed back into the incubator overnight. The next day the media was changed with complete culture media and again two days after. Cultures were monitored by eye till they reached 100% confluence, ⁇ 6-7 days after seeding.
  • the developed in vitro BBB model had high transendothelial electrical resistance (>100 ohms-cm 2 ) , measured using Millicell-ERS (Millipore, Billercia, MA) and very low Na-Fluorescein permeability (about 1-5 X 10 ⁇ 6 cm/s) .
  • 25 ⁇ g of purified peptide-AP fusions were added to the top chamber of the in vitro BBB model and transcytosed peptide-AP fusions were detected at 15 and 30 minute timepoints in the bottom chamber using ELISA. Briefly, 75 ⁇ of each sample was transferred to plates coated with 5 ]ig/ml mouse monoclonal anti-bacterial AP antibody (Sigma, St.

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Abstract

L'invention concerne des polypeptides de liaison au récepteur du facteur 1 de croissance de type insuline et des polynucléotides codant pour les polypeptides. L'invention concerne également des procédés d'obtention des polypeptides à l'aide de systèmes d'expression cellulaire et de systèmes exempts de cellules, et des procédés d'utilisation des polypeptides pour l'obtention de protéines hybrides. Les polypeptides de l'invention peuvent fournir un moyen d'administration d'agents thérapeutiques à travers la barrière hémato-encéphalique (BHE).
PCT/US2011/037904 2010-05-27 2011-05-25 Peptides de liaison au récepteur du facteur 1 de croissance de type insuline Ceased WO2011150061A1 (fr)

Priority Applications (23)

Application Number Priority Date Filing Date Title
SG2012086617A SG186068A1 (en) 2010-05-27 2011-05-25 Insulin-like growth factor 1 receptor binding peptides
BR112012030084A BR112012030084B1 (pt) 2010-05-27 2011-05-25 polipeptídeos que se ligam a receptores do fator de crescimento semelhante à insulina tipo 1, vetor, proteína de fusão e método in vitro para aplicação de um agente terapêutico através de células endoteliais
EA201291416A EA023541B1 (ru) 2010-05-27 2011-05-25 Пептиды, связывающиеся с рецептором инсулиноподобного фактора роста 1
UAA201214969A UA107596C2 (uk) 2010-05-27 2011-05-25 Виділений поліпептид, який зв'язується з рецептором інсуліноподібного фактора росту 1
DK11787324.0T DK2575846T3 (en) 2010-05-27 2011-05-25 Insulin-like growth factor-1 receptor binding peptides
KR1020127033486A KR101898502B1 (ko) 2010-05-27 2011-05-25 인슐린-유사 성장 인자 1 수용체 결합 펩티드
ES11787324.0T ES2550040T3 (es) 2010-05-27 2011-05-25 Péptidos de unión al receptor del factor 1 de crecimiento similar a la insulina
EP11787324.0A EP2575846B1 (fr) 2010-05-27 2011-05-25 Peptides de liaison au récepteur du facteur 1 de croissance de type insuline
MEP-2015-146A ME02206B (fr) 2010-05-27 2011-05-25 Peptides de liaison au récepteur du facteur 1 de croissance de type insuline
SI201130557T SI2575846T1 (sl) 2010-05-27 2011-05-25 Peptidi, ki vežejo receptor za inzulinu podoben rastni faktor 1
JP2013512202A JP5864556B2 (ja) 2010-05-27 2011-05-25 インスリン様成長因子１受容体結合ペプチド
RS20150591A RS54278B1 (sr) 2010-05-27 2011-05-25 Receptor insulinu-sličnog faktora rasta 1 vezujući peptidi
AU2011258301A AU2011258301B2 (en) 2010-05-27 2011-05-25 Insulin-like growth factor 1 receptor binding peptides
MX2012013710A MX337134B (es) 2010-05-27 2011-05-25 Peptidos de union al receptor del factor 1 de crecimiento similar a la insulina.
CA2800744A CA2800744C (fr) 2010-05-27 2011-05-25 Peptides de liaison au recepteur du facteur 1 de croissance de type insuline
HRP20151084TT HRP20151084T1 (hr) 2010-05-27 2011-05-25 Vezujuä†i peptidi receptora inzulinu sliäśnog faktora rasta 1
CN201180025866.2A CN103025341B (zh) 2010-05-27 2011-05-25 胰岛素样生长因子1受体结合肽
HK13110609.7A HK1183234B (en) 2010-05-27 2011-05-25 Insulin-like growth factor 1 receptor binding peptides
PL11787324T PL2575846T3 (pl) 2010-05-27 2011-05-25 Peptydy wiążące się z receptorem insulinopodobnego czynnika wzrostu 1
NZ603611A NZ603611A (en) 2010-05-27 2011-05-25 Insulin-like growth factor 1 receptor binding peptides
IL223030A IL223030A (en) 2010-05-27 2012-11-14 Receptor receptor-1 growth factor-1 insulin-like peptides
ZA2012/09689A ZA201209689B (en) 2010-05-27 2012-12-20 Insulin-like growth factor 1 receptor binding peptides
SM201500222T SMT201500222B (it) 2010-05-27 2015-09-22 Peptidi leganti il recettore del fattore di crescita 1 insulino-simile

Applications Claiming Priority (2)

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US34893710P 2010-05-27 2010-05-27
US61/348,937 2010-05-27

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WO2015131257A1 (fr) 2014-03-06 2015-09-11 National Research Council Of Canada Anticorps spécifiques du récepteur du facteur de croissance 1 analogues à l'insuline et leurs utilisations
WO2015131258A1 (fr) * 2014-03-06 2015-09-11 National Research Council Of Canada Anticorps spécifiques du récepteur du facteur de croissance 1 analogues à l'insuline et leurs utilisations
US10435436B2 (en) 2015-12-21 2019-10-08 Brainon Inc. Composition for improving memory, learning ability, and cognitive ability
US11369659B2 (en) 2015-12-21 2022-06-28 Brainon Inc. Method of enhancing a brain or cognitive function

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US10112998B2 (en) 2014-03-06 2018-10-30 National Research Council Of Canada Insulin-like growth factor 1 receptor-specific antibodies and uses thereof
EA035517B1 (ru) * 2014-03-06 2020-06-29 Нэшнл Рисеч Каунсил Оф Канада Антитела, специфические к рецептору инсулиноподобного фактора роста 1, и их применения
WO2015131258A1 (fr) * 2014-03-06 2015-09-11 National Research Council Of Canada Anticorps spécifiques du récepteur du facteur de croissance 1 analogues à l'insuline et leurs utilisations
KR20160127815A (ko) * 2014-03-06 2016-11-04 내셔날 리서치 카운실 오브 캐나다 인슐린-유사 성장 인자 1 수용체-특이적 항체 및 그의 용도
KR20160130436A (ko) * 2014-03-06 2016-11-11 내셔날 리서치 카운실 오브 캐나다 인슐린-유사 성장 인자 1 수용체-특이적 항체 및 그의 용도
KR20160130435A (ko) * 2014-03-06 2016-11-11 내셔날 리서치 카운실 오브 캐나다 인슐린-유사 성장 인자 1 수용체-특이적 항체 및 그의 용도
CN106459194A (zh) * 2014-03-06 2017-02-22 加拿大国家研究委员会 胰岛素样生长因子1受体特异性抗体及其用途
CN106536555A (zh) * 2014-03-06 2017-03-22 加拿大国家研究委员会 胰岛素样生长因子 1 受体特异性抗体及其用途
US10100117B2 (en) 2014-03-06 2018-10-16 National Research Council Of Canada Insulin-like growth factor 1 receptor-specific antibodies and uses thereof
US10106614B2 (en) 2014-03-06 2018-10-23 National Research Council Of Canada Insulin-like growth factor 1 receptor-specific antibodies and uses thereof
WO2015131257A1 (fr) 2014-03-06 2015-09-11 National Research Council Of Canada Anticorps spécifiques du récepteur du facteur de croissance 1 analogues à l'insuline et leurs utilisations
KR102355308B1 (ko) 2014-03-06 2022-01-24 내셔날 리서치 카운실 오브 캐나다 인슐린-유사 성장 인자 1 수용체-특이적 항체 및 그의 용도
WO2015131256A1 (fr) * 2014-03-06 2015-09-11 National Research Council Of Canada Anticorps analogues à l'insuline spécifiques du récepteur du facteur de croissance 1 et leurs utilisations
CN106536555B (zh) * 2014-03-06 2019-10-18 加拿大国家研究委员会 胰岛素样生长因子1受体特异性抗体及其用途
EA035472B1 (ru) * 2014-03-06 2020-06-22 Нэшнл Рисеч Каунсил Оф Канада Антитела, специфические к рецептору инсулиноподобного фактора роста 1, и их применения
EA035480B1 (ru) * 2014-03-06 2020-06-23 Нэшнл Рисеч Каунсил Оф Канада Антитела, специфические к рецептору инсулиноподобного фактора роста 1 и их применения
CN106459194B (zh) * 2014-03-06 2019-10-18 加拿大国家研究委员会 胰岛素样生长因子1受体特异性抗体及其用途
KR102355309B1 (ko) 2014-03-06 2022-01-24 내셔날 리서치 카운실 오브 캐나다 인슐린-유사 성장 인자 1 수용체-특이적 항체 및 그의 용도
KR102355310B1 (ko) 2014-03-06 2022-01-24 내셔날 리서치 카운실 오브 캐나다 인슐린-유사 성장 인자 1 수용체-특이적 항체 및 그의 용도
US10435436B2 (en) 2015-12-21 2019-10-08 Brainon Inc. Composition for improving memory, learning ability, and cognitive ability
US11369659B2 (en) 2015-12-21 2022-06-28 Brainon Inc. Method of enhancing a brain or cognitive function

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CY1116757T1 (el) 2017-03-15
CA2800744A1 (fr) 2011-12-01
MX2012013710A (es) 2013-05-20
RS54278B1 (sr) 2016-02-29
BR112012030084B1 (pt) 2020-04-14
HUE026269T2 (en) 2016-06-28
SG186068A1 (en) 2013-01-30
EP2575846B1 (fr) 2015-07-15
KR101898502B1 (ko) 2018-09-13
CN103025341B (zh) 2015-07-22
EA201291416A1 (ru) 2013-06-28
KR20130111237A (ko) 2013-10-10
AU2011258301B2 (en) 2013-11-21
AU2011258301A1 (en) 2012-12-06
HK1183234A1 (zh) 2013-12-20
NZ603611A (en) 2014-12-24
IL223030A (en) 2016-11-30
EP2575846A1 (fr) 2013-04-10
PT2575846E (pt) 2015-09-16
CA2800744C (fr) 2018-09-04
SMT201500222B (it) 2015-10-30
DK2575846T3 (en) 2015-08-10
ZA201209689B (en) 2014-05-28
JP5864556B2 (ja) 2016-02-17
JP2013534811A (ja) 2013-09-09
US20110294748A1 (en) 2011-12-01
ES2550040T3 (es) 2015-11-04
EP2575846A4 (fr) 2013-12-11
ME02206B (fr) 2016-02-20
CN103025341A (zh) 2013-04-03
US8415306B2 (en) 2013-04-09
SI2575846T1 (sl) 2015-09-30
IL223030A0 (en) 2013-02-03
EA023541B1 (ru) 2016-06-30
MX337134B (es) 2016-02-12
HRP20151084T1 (hr) 2015-11-06
BR112012030084A2 (pt) 2017-06-20
PL2575846T3 (pl) 2015-12-31

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